Assembly of the MexAB-OprM Multidrug Pump of Pseudomonas aeruginosa : Component Interactions Defined by the Study of Pump Mutant Suppressors

Abstract

In an effort to identify key domains of the Pseudomonas aeruginosa MexAB-OprM drug efflux system involved in component interactions, extragenic suppressors of various inactivating mutations in individual pump constituents were isolated and studied. The multidrug hypersusceptibility of P. aeruginosa expressing MexB with a mutation in a region of the protein implicated in oligomerization (G220S) was suppressed by mutations in the ␣/␤ domain of MexA. MexB(G220S) showed a reduced ability to bind MexA in vivo while representative MexA suppressors (V66M and V259F) restored the MexA-MexB interaction. Interestingly, these suppressors also restored resistance in P. aeruginosa expressing OprM proteins with mutations at the proximal (periplasmic) tip of OprM that is predicted to interact with MexB, suggesting that these suppressors generally overcame defects in MexA-MexB and MexB-OprM interaction. The multidrug hypersusceptibility arising from a mutation in the helical hairpin of MexA implicated in OprM interaction (V129M) was suppressed by mutations (T198I and F439I) in the periplasmic ␣-helical barrel of OprM. Again, the MexA mutation compromised an in vivo interaction with OprM that was restored by the T198I and F439I substitutions in OprM, consistent with the hairpin domain mediating MexA binding to this region of OprM. Interestingly, these OprM suppressor mutations restored multidrug resistance in P. aeruginosa expressing MexB(G220S). Finally, the oprM(T198I) suppressor mutation enhanced the yields of all three constituents of a MexA-MexB-OprM(T198I) pump as detected in whole-cell extracts. These data highlight the importance of MexA and interactions with this adapter in promoting MexAB-OprM pump assembly and in stabilizing the pump complex.Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an innate resistance to multiple antimicrobials (10), resistance increasingly attributable to the operation of broadly specific, multidrug efflux systems of the resistance-nodulation-division (RND) family (33, 34). Several RND family multidrug efflux systems have been described in P. aeruginosa (34, 35) although the major system contributing to intrinsic multidrug resistance is encoded by the mexAB-oprM operon (19). This system also contributes to acquired multidrug resistance as a result of its hyperexpression in nalB (i.e., mexR) (45, 53), nalC (6, 21), and nalD (42) mutants. The MexAB-OprM efflux system, like other tripartite RND family pumps, consists of an inner membrane drug-proton antiporter (the RND component; MexB), an outer membrane (OM) channel-forming component (also called outer membrane factor [OMF]; OprM) and a periplasmic membrane fusion protein ([MFP]; MexA) (32, 52).Crystal structures have now been reported for MexA (2, 11) and OprM (1), with the OprM structure reminiscent of that of TolC, the homologous OM component of the Escherichia coli AcrAB-TolC multidrug efflux system (18). The OprM channel is trimeric and is comprised of an OM-spanning ␤-barrel and a periplasmic ␣-helical barrel, wi...

Molecular insights into type I secretion systems

“…In vivo experiments showed that AcrA, MexA and HlyD can be cross-linked, resulting in the detection of dimers or trimers (Zgurskaya and Nikaido, 2000;Balakrishnan et al, 2001;Nehme and Poole, 2007;Zgurskaya et al, 2009). These results were obtained in the absence of the transporter and TolC .…”

The C-Terminal Domain of AcrA Is Essential for the Assembly and Function of the Multidrug Efflux Pump AcrAB-TolC

“…5). Mutagenesis studies support this interaction: the suppressor mutations in MexA that either restore the activity of the defective MexB or mutations in AcrA that enable the functional complex with MexB all map to the ␣-␤-barrel region of MFPs (11,17). However, in all these models, the C-domain of AcrA does not contribute to interactions with TolC and AcrB, and the assembly of the AcrABTolC complex does not require any significant changes in AcrA structure.…”

“…The replacement of aa 290 to 357 of AcrA with an analogous region of YhiU disrupted AcrA function possibly because of the loss of interaction with the AcrB transporter (5). Random mutagenesis of MexA identified C-terminal amino acid residues as important for MexA oligomerization and interaction with MexB (16,17).…”

Efflux-Mediated Drug Resistance in Bacteria

“…[113–118] AcrA drives TolC to fit the transporter complex. [119] Chimeric analysis of AcrA function reveals the importance of its C-terminal domain in its interaction with the AcrB pump.…”

“…[120] Mutations at both N- and C-terminus of MexA compromise the MexAB-OprM efflux activity, with the N-terminus involved in oligomerization of MexA and/or interaction with OprM and the C-terminus in interaction with the transporter MexB. [115, 118, 121] Construction of the chimeric, functional AcrA-MexB-TolC complex has suggested a certain degree of flexibility in accommodation. [122] In addition, there are paired MFPs as shown with TriAB of P. aeruginosa , which are both essential for TriABC-mediated triclosan resistance.…”